28 February 2023 Episode 319 Introduction to bird flu

Tue, 02/28/2023 - 15:04

Episode 319 Introduction to Avian Influenza Virus and outlook for a vaccine, aiming to cover bird flu

Dear colleagues,

As you know, there is quite some concern that, after COVID, a new respiratory pandemic could be based on Influenza A Virus (IAV) and more particularly on Avian Influenza Virus (AIV).  There is a booming literature and in this episode includes a general introduction, followed by some emerging vaccine examples.


Par 1 General introduction

Ep 319-1: Nino Rcheulishvili  Front Immunol Dec 2022 : the bigger picture


There are 18 different subtypes of HA and 11 different subtypes of NA amongst Influenza A meaning that there are potentially 198 subtypes of IAV out of which 131 have already been detected in nature.

All known IAV subtypes are found in birds except for H17N10 and H18N11 which have only been detected in bats. The low pathogenic AIV (LPAIV) and HPAIV do not cause any symptoms in wild birds

while HPAIV is lethal for domesticated birds.




Clearly H5Nx, H7Nx and H9Nx are considered high pathogenic




Most of the IAVs, including pandemic H1, H2, and H3 subtypes could be isolated from healthy poultry, meaning that the poultry remained without manifestation of any clinical signs.


H5N1– the most well-known strain of HPAIV was first isolated from a farmed goose in Guangdong Province, China in 1996. The first human infection of H5N1 in Hong Kong in 1997 and between 2003-2022 there have been 865 cases of human infection with H5N1 with a ~60% mortality rate from the live poultry markets while the poultry itself remained apparently healthy. 


H7N9 was transmitted in 2013 and caused 1,568 confirmed human cases where 616 (40 %) were fatal.



This figure illustrates the classical “antigenic drift” (point mutations mainly in HA and NA within a particular HN type) and “antigenic shift” (rearrangements of different HA and NA within the pig as “mixing vessel”).  In the upper part of B, it shows also another form of “antigenic shift”, where the HN combination remains the same, but where an accumulation of mutations allows for the bird flu prototype H1N1 to infect humans either directly or indirectly (via the pig). It is now hypothesized that the 1918 H1N1 pandemic originated from birds in this way.


Overview of 20th century flu pandemics



Following the 1918 Spanish flu, the next IAV pandemic emerged in 1957 via the spreading of H2N2 in Asia due to the reassortment between avian and human genes of the virus.

In 1968, H2N2 was followed by the H3N2 bird-human emergence which is called the Hong Kong pandemic

In 1977, H1N1 reemerged in Russia which was followed by the swine flu pandemic in 2009: the precursor gene (avian-human-swine) segments have circulated in pigs for over 10 years and, as a result H1N1/2009 was generated in swine via multiple reassortments over time.


This chronology demonstrates that it is only a matter of time before a future pandemic will occur.

It can be emerged by the accumulation of mutations or genetic reassortment.

Amongst the High Pathogenic Avian Influenza H5Nx and H7N9 are perceived to pose the highest danger.


Phylogenetic tree of IAV hemagglutinin (HA)




The 2 groups are colored cyan (group 1) and green (group 2), each of which can be further subdivided into 3 clades (H8, H9, and H12; H1, H2, H5, and H6; H11, H13, and H16) and 2 clades (H3, H4, and H14; H7, H10, and H15). (Rupert Russel Biochemistry 2008 https://doi.org/10.1073/pnas.0807142105)

The importance of the tree is that the groups and the clades or subtypes within these groups (see below) should be taken into account when trying to develop a “universal vaccine”


Nachbagauer Nat Med 2021 https://doi.org/10.1038/s41591-020-1118-7


Par 2 Examples of attempts for a broad vaccine, potentially addressing “bird flu”


This paragraph is by no means exhaustive.  I discuss just three papers:

  • Ep 319-2 with “classical” inactivated or live attenuated virus that have been engineered to elicit broad responses by focusing on hemagglutinin (HA): several variable “heads” combined with the H1 conserved “stalk”
  • Ep 319-3 including the HA stalk + two more conserved surface proteins (neuraminidase and M2)  + conserved nucleoprotein in a “simple” mRNA format, which is short-lived
  • Ep 319-4  prolonging the stimulation of H + N by including them in a self-amplifying RNA   


In all these papers the AIV-derived H5 is either included in the immunogen and/or used in the evaluation of immunogenicity or protection, with promising results.


Ep 319-2: Nachbagauer Nat Med 2021: Induction of broad cross-neutralization of group 1 IAV in humans by inactivated influenza virus, bearing chimeric HA. “Mosaic” approach.


The principle is to target group 1 HA by vaccinating consecutively with viruses that express H8 and H5 heads with H1 stalk in order to induce broad immunity.  The human subjects were chosen to have pre-existing immunity to H1 by natural exposure. 


The immunizing virus was either inactivated (IIV8 and IIV5) with AS03 as the adjuvant, compared with live-attenuated  influenza virus (LAIV5) with or without AS03. 



The figures in the results are a bit difficult to read, but the bottom lines are:


  • Induction of high titers of anti-stalk antibodies (the stalk is more conserved than the head).
  • Induction of broad antibodies against the “heads” of group 1, including H2, H9 and H18, but NOT against group 2 (H3).
  • The inactivated AS03 adjuvanted vaccine induces higher titers more rapidly than the live-attenuated vaccines
  • Passive transfer of serum from vaccinated humans in to mice protected against weight loss of the animals after infection with a group 1 IAV (H6)



The aim of the authors is to develop a similar “mosaic” strategy for group 2 IAV and for Influenza B virus into a trivalent vaccine (that) may enable protection against all drifted seasonal, zoonotic and emerging pandemic influenza viruses


Ep 319-3: Alec Freyn Mol Ther 2020: A Multi-Targeting, Nucleoside-Modified mRNA Influenza Virus Vaccine

Provides Broad Protection in Mice.


Principle: a lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccine to intradermally deliver a combination of conserved influenza virus antigens:

HA = hemagglutinin stalk, NA= neuraminidase,  M2 = matrix-2 ion channel, and NO = nucleoprotein.


Reminder of influenza structure                                              Vaccine construct



(A) Schematic representation of the mRNA-lipid nanoparticle vaccine technology that incorporates a 1-methylpseudouridine-modified mRNA molecule into an 80 lipid nanometer vesicle for efficient delivery into host cells upon vaccination. = very similar to Pfizer and Moderna vaccines.

(B) Diagrams illustrating the antigens used as immunogens in this study. Amino acid numbers are included under the mRNA coding for each antigen.


A single immunization with the combined mRNA construct protects mice from heterologous challenge within group 1 IAV




As can be seen there is partial protection with single mRNA (mini-HA, NA, M2 and NP) against body weight loss after challeng, but almost complete protection with the combined mRNA.


Ep 319-4: Cheng Chang Mol Ther 2022 Self-amplifying mRNA bicistronic influenza vaccines raise cross-reactive immune responses in mice and prevent infection in ferrets


The construct with a 5’ untranslated region (5’UTR), the replicase complex (NSP1-4) from Venezuelan Equine Encephalitis Virus (VEEV), one or two subgenomic promoters (SGP) in front of code for hemagglutinin (HA) end/or Neuraminidase (NA), followed by 3’ untranslated region (3’UTR) and  poly-adenosine (polyA) tail




In mice

  1. Self-amplifying (sa)-mRNA bicistronic A/H5N1 vaccines raised potent anti-HA and anti-NA neutralizing antibody responses and HA- or NA specific CD4+ and CD8+ T cell responses in mice  and boosted the cross-neutralizing response to heterologous A/H1N1




Female BALB/c mice, 8–10 weeks old, were immunized (10 mice/group) on days 1 and 22 with bilateral 50 mL intramuscular injections in the rear quadriceps. Serum samples were obtained from bleed-outs of euthanized animals on day 43. Monocistronic and bicistronic sa-mRNA vaccines with different antigen orders were evaluated for their ability to induce anti-H5 neutralizing antibody responses by (A) hemagglutination inhibition (HAI) assay, (B) microneutralization (MN) assay



  1. Similar immunogenicity results were obtained for bicistronic seasonal A/H3N2 and B/Yamagata vaccines.





In ferrets: sa-mRNA bicistronic A/H1N1 vaccine fully protected lung from infection by homologous virus


Illustration:  recovery of  infectious IAV from the lungs of vaccinated and challenged ferrets



Female domestic ferrets (n = 6) were immunized twice, 3 weeks apart, with 5.0 mg (blue) or 0.5 mg (red) of sa-mRNA H1-N1, 5.0 mg of sa-mRNA H1 (green) or sa-mRNA N1 (purple), or PBS (black) as control.

Ferrets were challenged 4 weeks after the second dose with A/Netherland/602/2009 (H1N1) virus at 106 50% tissue culture infectious dose

(TCID50) per animal and killed 4 days later.













According to a press release (Ep 319-5), this type of sa-RNA vaccine is now in phase 2 development to address “bird flu”  H5


CSL Seqirus announced in 2022 that the U.S. Biomedical Advanced Research and Development Authority (BARDA) selected the Massachusetts-based company to deliver anH5N8 A/Astrakhan3212/2020 (H5N8) virus vaccine candidate for assessment in a Phase 2 clinical study that is anticipated to begin in 2023. Under the $30.1 million agreement, BARDA is partnering with GSK and CSL Seqirus to manufacture investigational lots of H5N8 vaccine sand clinically assess the safety, immunogenicity, and dose-sparing ability of adjuvants in combination with the manufactured vaccine candidates.


The European Commission signed a framework contract on July 28, 2022, for the joint procurement of GSK's Adjupanrix, a pandemic influenza vaccine, based on H5N1 (Ep 319-6).  As a result, EC Member States can purchase up to 85 million vaccine doses, if necessary, during an influenza pandemic.  


I hope this introduction on bird flu was useful….  


Best wishes